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13 Cards in this Set
- Front
- Back
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What are human methyl reader proteins? What do they bind? How can these interacting be observed? |
-Class of over 200 proteins that all bind to methylated lysines or arginines -use protein microarray, treat potential targets with drug-biotin then streptavidin-dye |
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Advantages of microarrrays for drug binding? Disadvantages? |
Ad: -specific results are easy to interpret -good for studying classes of drug targets -lots of kinds of molecules can be arrayed Dis: - not the whole proteome -need a hypothetical target -biased method |
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Typical protocol for seeing what a drug binds? |
-Solid link chemical at point that is pointing out of pocket if possible -treat with cell lysate, with and without free inhibitor - analyze: SDS-PAGE followed by LC-MS/MS or other protein ID method |
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Example of converting chemical agents to affinity reagents? |
-Trapoxin can be made into K-trap matrix -observe what K-trap pulls down from lysate when free trapoxin is and isnt present -when bands disappear, it means they are specific binders. Remaining bands just happen to get stuck to beads no matter the test |
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Typical photoaffinity probes? What does UV light do to each? |
1. Benzophenone: forms ketyl radical 2. Phenyl azide: loss of N2, forms nitrene (terminal N with 2 lone pairs/very unstable) 3. Photoleucine: loss of N2, forms carbene, similar to nitrene |
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How can compounds be isolated by use of a photoaffinity probe? |
Derivatize probe with terminal alkyne, get probe to bind, irradiate with uv light (colavent bond forms), add biotin via click chemistry with N3, run over avidin coated beads |
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Old method of measuring cell proliferation? |
1. treat cell with BrDU 2. Allow cells to grow (BrdU takes place of T in DNA) 3. lyse/fix cells 4. denature to release DNA 5. Add anti-BrdU antibody (labelled) 6. Measure resulting bulk signal |
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New method for measuring cell proliferation? |
1. Treat cell with EdU 2. Allow cells to grow (EdU takes place of T) 3. add dye-azide (click rxn) 4. Measure resulting bulk signal |
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Advantage of new method? Disadvantage? |
Ad: do no need to lyse or denature DNA, so you can use a microscope to visualize newly made DNA Dis: you need copper, and too much copper can lead to cell death |
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How can glycoproteins be isolated? Visualized? |
-azido labelled sugars are incorporated into glycoproteins by native biosynthesis, thus can easily click on biotin (for pulldown) or dye (for microscopic analysis) |
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Method by which tumor growth can be imaged in live animals? |
1. Inject tumor cells into mouse 2. Treat mouse with azide linked to tumor targeting conjugate 3. treat mouse with azide reactive fluorescent dye (DBCO-Cy5) 4. Infrared fluorescence imaging reveals active tumor growth |
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What is needed for the incorporation of an unnatural amino acid? |
- a codon that is unused (stop codon) - AA-tRNA synthetase that's orthogonal to all other AAs and tRNAs |
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Why does the genetic code work? What therefore must be done for UAAs? |
- because aminoacyl tRNA synthetases load each AA onto its cognate tRNA -find or evolve an aminoacyl tRNA synthetase to charge your UAA onto a stop codon |
